Adrenomedullin agonists

ABSTRACT

A method for promoting bone growth in a patient (e.g., a mammal such as a human) said method including the step of administering a therapeutically effective amount of adrenomedullin or an adrenomedullin agonist to said patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/280,501, now U.S. Pat. No. 6,440,421, filed Mar. 30, 1999, which is acontinuation in part of 08/634,562, filed Apr. 18, 1996 now U.S. Pat.No. 5,888,963.

FIELD OF THE INVENTION

This invention relates to a method of promoting bone growth in apatient.

BACKGROUND OF THE INVENTION

Adrenomedullin is a 52-amino acid peptide originally identified in ahuman pheochromocytoma. It has since been found in normal adrenalmedulla, as well as other tissues such as the atria, ventricles,endothelial cells, lungs, brain and kidneys. Adrenomedullin alsocirculates in picomolar concentrations in both rats and man.

Several biological effects have been attributed to adrenomedullin. Ithas been described as a potent vasodilator, acting directly on therenal, cerebral, mesenteric, pulmonary, and systemic circulations.Administration of adrenomedullin can result in hypotension.

Binding of adrenomedullin to renal tubular membranes has also beenobserved, and sodium, potassium, and water excretion are increased byadrenomedullin. Adrenomedullin can in addition be a bronchodilator, andit has been reported to modulate the release of pituitary and vasoactivehormones.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that adrenomedullin andpeptide fragments of adrenomedullin can stimulate proliferation ofosteoblasts, which give rise to bone tissue. Accordingly, the inventionprovides methods for promoting bone growth, and compositions useful forpromoting bone growth.

In one aspect the invention provides a method for promoting bone growthin a patient, e.g., a mammal such as a human. The method includes thestep of administering a therapeutically effective amount ofadrenomedullin or an adrenomedullin agonist to the patient. The patientmay be suffering from a disease associated with excessive resorption orbreakdown of bone tissue such as osteoporosis or Paget's disease. Thepatient may also be suffering from bone losses as a result ofimmobility, bone fractures, malignancy, endocrine disorders, autoimmunearthritis, or drug use. The patient may also be undergoing a treatment(e.g., corticosteroid treatment, bone marrow transplantation, oroophorectomy) known to result in bone loss.

In another aspect, the invention features a method for prevent orretarding the development of a bone-associated diseases in a subject,e.g. a mammalian subject such as a human. Thus, the adrenomedullin andadrenomedullin agonists described herein may be used, e.g., to preventdevelopment of osteoporosis or Paget's disease in a subject.

A therapeutically effective amount depends upon the condition beingtreated, the route of administration chosen, and the specific activityof the compound used and ultimately will be decided by the attendingphysician or veterinarian. In one embodiment, the adrenomedullin agonistis administered to the patient until the patient's bone mass has beenrestored to normal levels. Thus, the duration of the administration maybe dependent upon the severity of the patient's bone loss.

The adrenomedullin or adrenomedullin agonist may be administeredparenterally, e.g., administered intravenously, subcutaneously, or byimplantation of a sustained release formulation. However, it will bereadily appreciated by those skilled in the art that the route, such asintravenous, subcutaneous, intramuscular, intraperitoneal, enterally,transdermally, transmucously, sustained released polymer compositions(e.g., a lactide polymer or copolymer microparticle or implant),profusion, pulmonary (e.g., inhalation), nasal, oral, etc., will varywith the condition being treated and the activity and bioavailability ofthe adrenomedullin or adrenomedullin agonist being used.

While it is possible for the adrenomedullin or adrenomedullin agonist tobe administered as the pure or substantially pure compound, it may alsobe presented as a pharmaceutical formulation or preparation. Theformulations to be used in the present invention, for both humans andanimals, comprise any of the adrenomedullin or adrenomedullin agoniststo be described below, together with one or more pharmaceuticallyacceptable carriers thereof, and optionally other therapeuticingredients.

The carrier must be “acceptable” in the sense of being compatible withthe active ingredient(s) of the formulation (e.g., capable ofstabilizing peptides) and not deleterious to the subject to be treated.Desirably, the formulation should not include oxidizing agents or othersubstances with which peptides are known to be incompatible. Forexample, adrenomedullin or adrenomedullin agonists in the cyclized form(e.g., internal cysteine disulfide bond) are oxidized; thus, thepresence of reducing agents as excipients could lead to an opening ofthe cysteine disulfide bridge. On the other hand, highly oxidativeconditions can lead to the formation of cysteine sulfoxide and to theoxidation of tryptophane. Consequently, it is important to carefullyselect the excipient. Ph is another key factor, and it may be necessaryto buffer the product under slightly acidic conditions (Ph 5 to 0.6).

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient(s) intoassociation with the carrier which constitutes one or more accessoryingredients.

In general, the formulations for tablets or powders are prepared byuniformly and intimately blending the active ingredient with finelydivided solid carriers, and then, if necessary, as in the case oftablets, forming the product into the desired shape and size.

Formulations suitable for parenteral (e.g., intravenous) administration,on the other hand, conveniently comprise sterile aqueous solutions ofthe active ingredient(s). Preferably, the solutions are isotonic withthe blood of the subject to be treated. Such formulations may beconveniently prepared by dissolving solid active ingredient(s) in waterto produce an aqueous solution, and rendering said solution sterile. Theformulation may be presented in unit or multi-dose containers, forexample, sealed ampoules or vials.

Formulations suitable for sustained release parenteral administrations(e.g., biodegradable polymer formulations) are also well known in theart. See, e.g., U.S. Pat. Nos. 3,773,919 and 4,767,628 and PCTPublication No. WO 94/15587.

The adrenomedullin or adrenomedullin agonist may also be administeredwith an bone anti-resorptive agent or another bone anabolic agent.Examples of bone anti-resorptive agents include calcitonin,bisphosphonates (e.g., etidronate, alendronate, or pamidronate),estrogen, and analogs thereof. Examples of bone anabolic agents includeparathyroid hormone, parathyroid hormone related protein, cytokines(e.g., TGF-β, IGF-1), growth hormone, and analogs thereof.

In another aspect, the invention features a peptide consisting ofbetween 30 and 26 amino acids and comprising the sequenceadrenomedullin(27-52) (SEQ ID NO:1) wherein the carboxyl terminal is afree acid or amidated. Examples of such peptides includeadrenomedullin(27-52) (SEQ ID NO: 1); adrenomedullin(26-52) (SEQ IDNO:2); adrenomedullin(25-52) (SEQ ID NO:3); adrenomedullin(24-52) (SEQlID NO:4); or adrenomedullin(23-52) (SEQ ID NO:5) wherein the carboxylterminal is amidated. Peptides of this invention are described herein,for example, by the following format: adrenomedullin(13-52) (SEQ ID NO:11). The numbers between the parentheses refer to the number of aminoacids present in the peptide, e.g., the forty amino acid fragmentbetween the seine residue at position 13 and the amidated tyrosineresidue at position 52 of adrenomedullin. The sequence of adrenomedullin(SEG ID NO:18) is listed in FIG. 1 of European Patent Application No.622,458 A2.

The invention also includes an adrenomedullin (ADM) agonist 5-25, 6-25or 7-25 amino acids in length. The ADM agonist comprises a fraction orthe entirety of the amino acid sequence ADM(32-40). In some embodimentsthe ADM agonist is 9-25 amino acids in length and includes the aminoacid sequence ADM(32-40), e.g., the ADM agonist can include the aminoacid sequences ADM (28-40), ADM(32-52), and ADM (30-52). Other examplesof ADM agonists include peptides 10-25, 12-22, or 13-23 amino acids inlength and containing the amino acid sequence ADM(32-40).

The ADM agonists described herein are useful in treating diseases forwhich increased osteoblast proliferation is desirable. Thus, the ADMagonists are useful for treating bone-associated conditions or diseasessuch as osteoporosis and Paget's disease and in preventing or inhibitingthe development of such bone-associated diseases in subjects at risk fordeveloping these diseases.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

It is believed that one skilled in the art can, based on the descriptionherein, utilize the present invention to its fullest extent. Thefollowing specific embodiments are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Also, all publications, patentapplications, patents, and other references mentioned herein areincorporated by reference.

Adrenomedullin and Adrenomedullin Agonists

Adrenomedullin is a 52-amino acid having an amidated C-terminus. Manyanalogs, e.g., carboxyl terminal fragments, have been prepared, such asadrenomedullin(15-52), adrenomedullin(1-50), adrenomedullin(11-50),adrenomedullin(22-52), adrenomedullin(40-52), adrenomedullin(13-52),adrenomedullin(47-52), and adrenomedullin(45-52). See European PatentNo. 622,458 A2; Lin, et al., Eur. J. Pharmacol. 260:1-4 (1990); andSantiago, J. A., et al., Eur. J. of Pharmacology 272:115-118 (1995).What is meant by an adrenomedullin agonist is a compound which (1) has ahigh affinity (e.g., a Ki of less than 1 μM) for the adrenomedullinreceptor (as defined by the receptor binding assay described in Owji, etal., Endocrinology 136(5):2128 (1995)) and (2) promotes theproliferation of osteoblast cells (as defined below in the osteoblastproliferation assays).

Synthesis

The synthesis of short amino acid sequences is well established in thepeptide art. See, e.g., Stewart, et al., Solid Phase Peptide Synthesis(2d ed., 1984). The following is the synthesis of adrenomedullin(27-52). Other such adrenomedullin agonists can be prepared by makingappropriate modifications, within the ability of someone of ordinaryskill in the field of the synthetic method described herein.

Benzhydrylamine-polystyrene resin (Advanced ChemTech, Inc., Louisville,Ky.) (0.6 g, 0.25 mmole) in the chloride ion form was placed in thereaction vessel of an Advanced ChemTech peptide synthesizer programmedto perform the following reaction cycle: (a) methylene chloride; (b) 33%trifluoroacetic acid in methylene chloride (2 times for 1 and 15 mineach); (c) methylene chloride; (d) ethanol; (e) methylene chloride; and(f) 10% diisopropylethylamine in methylene chloride.

The neutralized resin was stirred with Boc-Tyr(2,6-dichlorobenzyl) anddiisopropylcarbodiimide (0.75 mmole each) in methylene chloride for 1 hand the resulting amino acid resin was then cycled through steps (a) to(f) in the above wash program. The following amino acids (0.75 mmole)were then coupled successively by the same procedure: Boc-Gly, Boc-Gln,Boc-Pro, Boc-Ser(benzyl), Boc-Ile, Boc-Lys(2-chloro-CBZ),Boc-Ser(benzyl), Boc-Arg(p-Tosyl), Boc-Pro, Boc-Ala, Boc-Ala, Boc-Val,Boc-Asn, Boc-Asp(cyclohexyl), Boc-Lys(2-chloro-CBZ),Boc-Asp(cyclohexyl), Boc-Lys(2-chloro-CBZ), Boc-Asp(cyclohexyl),Boc-Thr(benzyl), Boc-Phe, Boc-Gln, Boc-Tyr(2,6-dichlorobenzyl), Boc-Ile,Boc-Gln, Boc-His (benzyloxymethyl), and Boc-Ala. After removal of thelast Boc group and washing and drying, the completed resin weighed 1.11g. All amino acids were purchased from Bachem California, (Torrence,Calif.).

The resin described above (1.11 g, 0.25 mmole) was mixed with anisole (5ml), dithiothreitol (100 mg) and anhydrous hydrogen fluoride (35 ml) atOUC and stirred for 45 min. Excess hydrogen fluoride was evaporatedrapidly under a stream of dry nitrogen, and free peptide precipitatedand was washed with ether. The crude peptide was then dissolved in aminimum volume of 2 M acetic acid and eluted on a column (2.5×100 cm) ofSephadex G-50 (Pharmacia, Piscataway, N.J.) using the same solvent.Fractions containing a major component, detected by ultravioletabsorption and thin layer chromatography, were then pooled, evaporatedto a small volume, and applied to a column (2.5×50 cm) of Vydacoctadecylsilane silica (10-15μ; Rainin, Emeryville, Calif.). This columnwas eluted with a linear gradient of 10-45% acetonitrile in 0.1%trifluoroacetic acid in water. Fractions were examined by thin layerchromatography (tlc) and analytical high performance liquidchromatography (hplc) and pooled to give maximum purity. Repeatedlyophilization of the solution from water gave the product as the amideand as a white, fluffy power. The product is found to be homogeneous byhplc and tlc. Amino acid analysis of an acid hydrolysate confirmed thecomposition of the peptide. Laser desorption mass spectroscopy gave theexpected molecular weight.

In vitro Studies

Adrenomedullin agonists can be tested for their ability to promote bonegrowth by examining their activity in the in vitro and in vivo assaysdescribed herein.

Osteoblast Cell Culture

Osteoblasts were isolated by collagenase digestion from 20-day fetal ratcalvariae. The calvariae were then treated twice with phosphate bufferedsaline (PBS; Gibco, Grand Island, N.Y.) containing 3 Mm ethylenediamine-acetic acid (EDTA; Sigma Chemical Co., St. Louis, Mo.) for 15minutes at 37

C. in a shaking water bath. After washing once in PBS, 3 ml of 1 mg/mlcollagenase (Sigma Chemical Co.) was added to the calvariae for 10minutes at 37

C. After discarding the supernatant, the calvariae were treated twicewith 2 mg/ml collagenase (30 mins, 37

C.). The supernatants were then centrifuged and the cells washed inDulbecco's Modified Eagle's Medium (DMEM; Gibco Labs, Grand Island,N.Y.) with 10% fetal calf serum (FCS), suspended in further DMEM/10%FCS, and placed in 75 cm³ flasks. The cells were incubated under 5% CO₂and 95% air at 37

C.

The osteoblast-like character of these cells has been established bydemonstration of high levels of alkaline phosphatase and osteocalcin,and a sensitive adenylate cyclase response to parathyroid hormone andprostaglandins. Confluence was reached by 5-6 days, at which time thecells were subcultured. After trypsinization using trypsin-EDTA(0.05%/0.53 Nm; Gibco Labs, Grand Island, N.Y.), the cells were rinsedin Minimal Essential Medium (MEM; Gibco Labs) with 10% FCS andresuspended in MEM with 5% FCS, then seeded (10⁵ cells/ml) in 24-wellplates (0.5 ml cell suspension per well, i.e., 5×10⁴ cells/well).

Proliferation Assays

Cell proliferation studies (i.e., cell counts and thymidineincorporation) were performed both in actively-growing andgrowth-arrested cell populations. To produce actively-growing cells,subconfluent populations (24 hrs after subculturing) were changed tofresh MEM which contained 1% FCS and the test compounds. To producegrowth-arrested cells, subconfluent populations were changed toserum-free medium with 0.1% bovine serum albumin plus the testcompounds.

A. Cell Counts:

The effect of adrenomedullin on proliferation of fetal ratosteoblast-like cells was first assessed by the measurement of cellnumbers. Cell counts were analyzed at 6, 24, and 48 hours after additionof the test compound or vehicle. The cell numbers were determined afterremoving cells from the wells by exposure to trypsin/EDTA (0.05%/0.53Mm) for 5 minutes at 37

C. Counting was performed in a hemocytometer chamber. Results wereexpressed per well.

Treatment with adrenomedullin for 24 hours, in cultures grown in mediumcontaining 1% FCS, produced a dose-dependent increase inactively-growing osteoblasts. A significant increase was observed atadrenomedullin concentrations of 10⁻¹²M and greater. This stimulationwas maintained for at least 48 hours.

To determine whether proliferation in response to adrenomedullin wasdependent on the basal growth rate of the cells, these experiments wererepeated in growth-arrested osteoblast preparations. The time-course ofthe increase in cell number in response to adrenomedullin (10⁻¹⁰M) inthese cells was similar to that seen in actively-growing cells.

Treatment for 24 hrs with the test compounds adrenomedullin(15-52),adrenomedullin(22-52), and adrenomedullin(27-52) produced similardegrees of proliferation to that of the full length adrenomedullinpolypeptide.

B. DNA Synthesis:

The effect of adrenomedullin on DNA synthesis in osteoblasts was thenassessed by the measurement of [³H]-thymidine incorporation intoisolated fetal rat osteoblast-like cells. [³H]-thymidine incorporationinto actively-growing and growth-arrested cells was assessed by pulsingthe cells with [³H]-thymidine (1 μCi/ml) (Amersham, Arlington Heights,Ill.) two hours before the end of the experimental incubation. Theexperiment was terminated at 6, 24, or 48 hours by washing the cells inMEM with cold thymidine followed by 10% trichloroacetic acid. Theprecipitate was washed twice with ethanol:ether (3:1), and the wellswere desiccated at room temperature. The residue was redissolved in 0.5M KOH at 85

C. for 30 mins, neutralized with 1 M Hcl, and an aliquot counted forradioactivity. Results were expressed as cpm per well.

Treatment with adrenomedullin (10⁻¹⁰M) for 24 hours significantlystimulated [³H]-thymidine incorporation into both actively-growing andgrowth-arrested cells.

The effect of adrenomedullin fragments on proliferation of primarycultures of osteoblasts was also tested. Addition of either ADM(28-40),ADM(32-52), ADM (28-40), and ADM (27-52) to a final concentration of 1nm produced a statistically significant increase in cell number relativeto control osteoblasts. Thymidine incorporation was also increased inosteoblasts treated with 1 nm of these fragments.

Bone Organ Culture

Bone resorption studies were carried out in neonatal mouse calvariae aspreviously described (Reid, et al., Endocrinology 126:1416-1420 (1990)).Mice were injected subcutaneously with 5 μCi ⁴⁵Ca (Amersham) at 2 daysof age, and hemi-calvariae were dissected out 4 days later.Hemi-calvariae were pre-incubated for 24 hours in medium 199 (Gibco BRL,Grand Island, N.Y.) with 0.1% bovine serum albumin, then changed tofresh medium containing adrenomedullin or control vehicle. Incubationwas continued for a further 48 hours. In the last 4 hours of incubation[³H]-thymidine was incorporated into the organ cultures as describedpreviously (Lowe, et al., Calif. Tissue Int. 49-394-397 (1991)). Therewere 5-7 calvariae in each group.

There was no significant change in ⁴⁵Ca release from prelabeledcalvariae treated for 48 hours with adrenomedullin at concentrations of10⁻⁷ M to 10⁻¹⁰ M indicating that adrenomedullin did not stimulate boneresorption. In contrast, [³H]-thymidine incorporation was significantlyincreased in the same experiments indicating that adrenomedullinstimulated bone growth.

In vivo Studies

The local effects of adrenomedullin on bone histology in vivo wereassessed in adult mice using a previously described model (Cornish etal., Endocrinology 132:1359-1366, 1993). Sexually mature male ARC SwissWebster mice aged between 40 and 50 days and weighing 25-35 g were giveninjections (25 μl) over the periosteum of the right hemicalvariae for 5consecutive days. Previous studies with dye injections have shown thatthe injected material spreads over much of the injected hemicalvariaebut does not cross the midline. The animals were maintained on a lowcalcium diet (0.1%) from 5 days prior to the first injection. Two groupsof mice (n=12 in each) were injected daily with adrenomedullin in dosesof 4×10⁻¹⁰ M and 4×10⁻⁹ M, respectively. These doses were chosen basedon experience with other bone-active peptides in this model. Animals inthe control group (n=16) were injected with vehicle (water) only. Allanimals were sacrificed 1 week following the last injection. The studyhad the approval of the local institutional review board.

The calvariae were dissected free of soft tissue, and gross morphologywas assessed by examination of the intact calvariae under a dissectionmicroscope. Bone tissue was fixed in 10% phosphate-buffered formalin,dehydrated in a graded series of ethanol solutions and embeddedundecalcified in methyl methacrylate resin. Sections (4 μm thick) werecut on a Leitz rotary microtome (Leica Instruments, Nussloch, Germany)using a tungsten-carbide knife, then mounted on gelatin-coated slidesand air-dried. The sections were stained with a Goldner tri-chrome stainand examined using an Olympus BX 50 microscope (Olympus Optical Co Ltd,Tokyo, Japan) which was attached to an Osteomeasure Image Analyzer(Osteometrics Inc., Atlanta, Ga.). Histomorphometric analyzes were madeof three adjacent fields (using a 20× objective) in each hemicalvaria.This results in measurements being made over >90% of the length of eachhemicalvaria.

The parameters assessed are as defined by the American Society for Boneand Mineral Research (Parfitt et al. J. Bone Miner. Res. 2:595-610,(1987)), and are expressed per mm of calvarial length. Osteoblasts weredefined as cells immediately adjacent to osteoid. Osteoclast numbersincluded only multinucleated cells. The various surface estimates werebased on measurements of both periosteal and intramembranous surfaces:those eroded by osteoclasts (eroded perimeter), those immediatelyadjacent to osteoclasts (osteoclast perimeter), and those immediatelyadjacent to osteoblasts (osteoblast perimeter). The precision of thesehistomorphometric measurements in these studies (expressed ascoefficients of variation of paired measurements) are as follows:mineralized bone area 1.3%, osteoid area 6.9%, osteoblast perimeter6.8%, osteoblast number 1.7%, eroded perimeter 6.7%, osteoclastperimeter 7.9%, osteoclast number <1.0%, and calvarial length 0.2%. Allmeasurements were made by one operator who was blinded to the treatmentgroup of each bone.

In the in vivo experiment, the primary end-point for eachhistomorphometric index was the determination of whether it wasdifferent in the injected right hemicalvaria from the contralateral,uninjected bone of the same animal. The absolute values of eachhistomorphometric index are presented for the injected and uninjectedhemicalvariae by treatment group in Tables 1-3. The tables set out thehistomorphometric indices in the uninjected and injected hemicalvariaefrom each of three groups: mice injected with vehicle (Table 1), miceinjected with 4×10⁻¹⁰ M adrenomedullin (Table 2), or mice injected with4×10⁻⁹ M adrenomedullin (Table 3). Statistical analysis performed on theratios of each index in the injected to the uninjected halves of eachcalvariae revealed two- to three-fold increases in the indices ofosteoblast activity in those bones exposed to either dose ofadrenomedullin. Resorption indices showed slight upward trends in thepresence of adrenomedullin but only for eroded perimeter was thissignificant. Mineralized bone area was increased by 13.6% with thehigher dose of adrenomedullin. Periosteal area was not changed (P=0.54).

Tables 1-3. Effects of Adrenomedullin on Histomorphometric Indices inAdult Mouse Calvariae in vivo*

TABLE 1 Vehicle Index Uninjected Injected Osteoid Area 0.0025 (0.0004)0.0021 (0.0003) Ob Perimeter 0.69 (0.10) 0.76 (0.07) Number of Obs 65(8) 68 (5) Eroded Pm 0.74 (0.10) 0.76 (0.10) Oc Pm 0.12 (0.01) 0.13(0.02) Number of Ocs 4.4 (0.3) 4.5 (0.3) Periosteal Area 0.014 (0.002)0.013 (0.001) Bone Area 0.113 (0.005) 0.106 (0.005)

TABLE 2 Adrenomedullin 4 × 10⁻¹⁰ mol Index Uninjected Injected OsteoidArea 0.0016 (0.0003) 0.0041 (0.0005) Ob Perimeter 0.72 (0.10) 1.21(0.13) Number of Obs 57 (5) 111 (11) Eroded Pm 0.77 (0.13) 0.85 (0.09)Oc Pm 0.12 (0.02) 0.14 (0.01) Number of Ocs 4.4 (0.4) 5.2 (0.3)Periosteal Area 0.013 (0.002) 0.015 (0.001) Bone Area 0.115 (0.005)0.122 (0.006)

TABLE 3 Adrenomedullin 4 × 10⁻⁹ mol Index Uninjected Injected OsteoidArea 0.0012 (0.0002) 0.0032 (0.0008) Ob Perimeter 0.56 (0.06) 1.33(0.09) Number of Obs 61 (5) 105 (6) Eroded Pm 0.67 (0.08) 0.67 (0.07) OcPm 0.12 (0.01) 0.15 (0.01) Number of Ocs 4.4 (0.3) 5.0 (0.3) PeriostealArea 0.011 (0.001) 0.012 (0.002) Bone Area 0.114 (0.006) 0.128 (0.007) *Data are mean (se). Ob, osteoblast; Pm, perimeter; Oc, osteoclast

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Forexample, the adrenomedullin agonists described herein may include aminoacids linked via non-peptidyl linkages, e.g., a peptide in which one ormore peptide bonds have a carbonyl oxygen replaced with a methylenegroup. The adrenomedullin agonists may alternatively or in additioninclude amino acids other than the encoded amino acids. These include,e.g., substitution of the naturally occurring L-form of an encoded aminoacid with its corresponding D-form. Other aspects, advantages, andmodifications are within the claims.

1. An adrenomedullin (ADM)(SEQ ID NO: 18) agonist 9-25 amino acids inlength, wherein said ADM agonist is a fragment of ADM which comprisesthe amino acid sequence ADM (32-40)(SEQ ID NO:14).
 2. The ADM agonist ofclaim 1, wherein the carboxyl terminus is amidated.
 3. The ADM agonistof claim 1, wherein said ADM agonist comprises the amino acid sequenceADM (28-40)(SEQ ID NO: 15).
 4. The ADM agonist of claim 1, wherein saidADM agonist comprises the amino acid sequence ADM (32-52)(SEQ ID NO:16).
 5. A pharmaceutical composition comprising the ADM agonist of claim1.